Huntington's Disease (HD) is the most common of polyglutamine expansion disorders, with many studies suggesting that pathogenesis involves a conformational change in the mutant protein, huntingtin, with gain of a deleterious function, possibly involving a proteolytic fragment (or fragments) from the N-terminal region (containing the expanded polyglutamine region) that leads to abnormal protein interactions and neuronal apoptotic death. Whether aggregates containing N-terminal fragments are the cause of toxicity or if they are generated as part of a cellular protective response remains a controversial and crucial question. To begin to address this, we have developed an inducible cell model system in rat PC12 cells (a neuronal-like cell line) with initial results that suggest that proteolytic release of N-terminal fragments from the full-length human huntingtin protein precedes toxicity, with a subset of these fragments translocating and forming aggregates in the nucleus, prior to cell death. We will construct and characterize additional inducible cell lines which express full-length mutant and normal Htt, with robust, moderate and low levels of expression, relative to the existing cell lines, to provide confirming evidence of these proteolytic events, and provide additional information concerning their relation to toxicity and aggregation. The second aim will confirm the hypothesis that proteolytic cleavage of the mutant huntingtin precedes cell death and test if higher levels of full length mutant huntingtin lead to higher levels of N- terminal fragments, leading to cell death sooner. This will be done using western analysis and flow cytometric apoptosis assays. The third aim will test the hypothesis that cell lines with robust expression of full-length mutant huntingtin will show a higher level of nuclear aggregation of N-terminal fragments leading to a higher level of cell death than the lines expressing huntingtin to a lesser extent. This will be done using indirect immunofluorescence assays concurrently with TUNEL assays for apoptosis. These cell lines will be the first inducible HD cell model system that expresses different amounts of the full-length mutant huntingtin in a neuronal-like environment. Not only will these be useful for studying the pathological mechanism leading to Huntington's disorder, but they will be used for future screening of potential therapeutics. PUBLIC HEALTH RELEVANCE: Huntington's disease (HD) is an adult onset autosomally dominant inherited neurodegenerative disease that manifests with overlapping cognitive, psychiatric and physical symptoms, ultimately resulting in premature death. About 30,000 people in the United States have been diagnosed with symptomatic HD, with another 250,000 to 300,000 people at-risk. There is currently no known cure and minimal effective symptomatic treatment. The cell lines constructed in this project will be the first inducible HD cell model system that expresses different amounts of the full length mutant huntingtin in a neuronal like environment. Characterization of these cell lines will advance understanding of the pathologic mechanism leading to the disease state and provide cell model systems to screen potential therapeutics in future studies.